OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1–6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

中文翻译：

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鉴定具有强效抗疟疾活性的一流疟原虫 OTU 抑制剂

OTU 蛋白酶拮抗宿主细胞中的细胞防御并参与发病机制。有趣的是，恶性疟原虫、间日疟原虫和约氏疟原虫具有未表征且高度保守的病毒 OTU 样蛋白。然而，它们的结构、功能或抑制剂以前没有报道过。为此，我们进行了结构建模、小分子筛选、解偶联分析，以表征和开发恶性疟原虫、间日疟原虫和约氏疟原虫 OTU 样蛋白的一流抑制剂。这些疟原虫 OTU 样蛋白在催化和抑制口袋中具有高度保守的残基，类似于病毒 OTU 蛋白。疟原虫 OTU 蛋白证明了泛素和 ISG15 解偶联活性，如通过蛋白质印迹和流式细胞术分析的细胞内泛素化蛋白含量所证明的那样。我们筛选了一个小分子库，以确定具有有效抗疟疾活性的疟原虫 OTU 抑制剂。富集和相关性研究确定了结构相似的分子。我们已经鉴定出两种小分子，它们可抑制恶性疟原虫、间日疟原虫和约氏疟原虫 OTU 蛋白（IC50 值低至 30 nM），具有有效的抗疟疾活性（IC50 为 4.1–6.5 µM）。我们还建立了酶动力学、药物相似性、ADME 和 QSAR 模型。MD 模拟使我们能够解决抑制剂如何与疟原虫 OTU 蛋白相互作用。这些发现表明，针对疟疾 OTU 样蛋白酶是开发新的抗疟疾疗法的可行策略。我们已经鉴定出两种小分子，它们可抑制恶性疟原虫、间日疟原虫和约氏疟原虫 OTU 蛋白（IC50 值低至 30 nM），具有有效的抗疟疾活性（IC50 为 4.1–6.5 µM）。我们还建立了酶动力学、药物相似性、ADME 和 QSAR 模型。MD 模拟使我们能够解决抑制剂如何与疟原虫 OTU 蛋白相互作用。这些发现表明，靶向疟疾 OTU 样蛋白酶是开发新抗疟疾疗法的合理策略。我们已经鉴定出两种小分子，它们可抑制恶性疟原虫、间日疟原虫和约氏疟原虫 OTU 蛋白（IC50 值低至 30 nM），具有有效的抗疟疾活性（IC50 为 4.1–6.5 µM）。我们还建立了酶动力学、药物相似性、ADME 和 QSAR 模型。MD 模拟使我们能够解决抑制剂如何与疟原虫 OTU 蛋白相互作用。这些发现表明，针对疟疾 OTU 样蛋白酶是开发新的抗疟疾疗法的可行策略。